Compositions For Treating Bacterial Infections

ABSTRACT

Polynucleotides encoding a mutant human carboxylesterase enzyme and polypeptides encoded by the polynucleotides which are capable of metabolizing a prodrug and inactive metabolites thereof to active drug are provided. Compositions and methods for sensitizing cells to a prodrug agent, inhibiting cell growth, treating drug addiction, and facilitating the metabolism of an organophosphate with this enzyme are also provided. In addition, a screening assay for identification of drugs activated by this enzyme is described.

This application is a divisional of U.S. Ser. No. 13/020,871 filed Feb.4, 2011, which is a divisional of U.S. Ser. No. 12/306,031 filed Jan.28, 2009, which is the U.S. National Phase of PCT/US2007/071640 filedJun. 20, 2007 which claims benefit of U.S. Provisional Ser. No.60/805,643, filed Jun. 23, 2006, the contents of which are incorporatedherein by reference in their entireties.

This invention was made with government support under grant numbersCA76202, CA79763, CA98468, CA108775, DA18116 awarded by NationalInstitutes of Health and grant number P30 CA 21765 awarded by CancerCenter Core. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel polynucleotides which encode a mutanthuman carboxylesterase enzyme, polypeptides encoded by thesepolynucleotides and vectors and host cells comprising these vectorswhich express the enzyme. This enzyme is capable of metabolizingchemotherapeutic prodrugs and inactive metabolites into active drug. Theinstant invention thus relates to compositions comprising thesepolynucleotides and methods for sensitizing selected cells to a prodrugby transfecting the cells with a polynucleotide placed under the controlof a disease-specific responsive promoter. Sensitized cells can then becontacted with a prodrug to alter the activity of the cell in somemanner, such as inhibition of tumor cell growth. Alternative methodscontemplated by the invention would apply to treatment of drug addictionand resistance to chemical weapons. The invention further includes noveldrug screening assays for identifying new prodrugs that are activated bythis enzyme.

2. Background of the Invention

Development of effective human anti-cancer drugs has often been hinderedby the inability of tested agents to selectively target tumor cells overnon-tumor cells. This lack of selectivity of many anti-cancer drugsoften leads to unwanted and often serious side effects in patients thatcan limit the dose of the drug administered. A goal of all human drugtherapies, regardless of the disease or condition being treated, is tobe able to administer the lowest dose of a drug that produces thedesired clinical effect. Therefore, strategies for more efficient drugdelivery are continuously sought.

In the case of certain anti-cancer drugs, one strategy has beendevelopment of prodrug forms of active compounds that can increase thebioavailability of the drug and increase its ability to effectively killtumor cells. One example of this prodrug strategy has been thedevelopment of carboxylesterase (CE) pro-drugs (Roosebaum, et al. 2004.Pharmacol. Rev. 56:53-102).

CEs are ubiquitous serine esterase enzymes that catalyze conversion ofcarboxylic esters to an alcohol and a carboxylic acid as well ashydrolyzing amides, thioesters, phosphoric acid esters and acidanhydrides. In some cases, CE enzyme activity is responsible for thedetoxification of xenobiotics. CEs are present in high levels in bothnormal and tumor tissue, especially in liver, kidney, testis, lung andplasma. In addition to their known ability to detoxify certainchemicals, recent research has focused on the ability of these enzymesto be used in design of prodrug forms of certain cytostatic drugs(Roosebaum, et al. 2004. Pharmacol. Rev. 56:53-102). Examples of theapplication of CE activity to prodrug development are CPT-11,paclitaxel-2-ethylcarbonate, and capecitabine. In all three of thesecases, CE activity leads to production of an active cytostatic drug,SN-38, paclitaxel, and 5′-DUFR, respectively. In addition, a particularhuman CE known as hCE1 has been shown to catalyze the hydrolysis ofcertain drugs of abuse, specifically heroin and cocaine, and to catalyzethe transesterification of cocaine in the presence of ethanol to itstoxic metabolite, cocaethylene (Redinbo, et al. 2003. Biochem. Soc.Trans. 31:620-624). hCE1 is also being developed by the United Statesmilitary as a prophylactic agent for treating potential exposures tochemical weapons such as the organophosphates Sarin, Soman, Tabun, andVX gas (Redinbo, et al. 2003. Biochem. Soc. Trans. 31:620-624).

CPT-11 (irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is aprodrug that has been investigated for the treatment of cancer, and isconverted to the active drug known as SN-38(7-ethyl-10-hydroxy-camptothecin) (Tsuji, et al. 1991. J. Pharmacobiol.Dynamics 14:341-349; Satoh, et al. 1994. Biol. Pharm. Bull. 17:662-664).SN-38 is a potent inhibitor of topoisomerase I (Tanizawa, et al. 1994.J. Natl. Cancer Inst. 86:836-842; Kawato, et al. 1991. Cancer Res.51:4187-4194), an enzyme whose inhibition in cells results in DNA damageand induction of apoptosis (Hsiang, et al. 1989. Cancer Res.49:5077-5082). In addition to metabolism to SN-38, in humans CPT-11 isalso metabolized to a compound known as APC (Haaz, et al. 1998. CancerRes. 58:468-472). APC has little, if any, anti-tumor activity and is notconverted to an active metabolite in humans (Rivory, et al. 1996. CancerRes. 56:3689-3694). CPT-11 has demonstrated remarkable anti-tumoractivity in pre-clinical models and Phase I/II clinical trials (Furman,et al. 1999. J. Clin. Oncol. 17:1815-1824; Houghton, et al. 1996. Clin.Cancer Res. 2:107-118; Houghton, et al. 1995. Cancer Hcemother.Pharmacol. 36:393-403), and as such is being tested against a variety ofhuman malignancies. However, myelosuppression and secretory diarrhealimit the amount of drug that can be administered to patients.Accordingly, before this promising anti-cancer agent can be usedsuccessfully, these dose-limiting toxicities must be overcome. CPT-11 iscurrently approved for use in human colon cancer.

The active anti-tumor agent of CPT-11, SN-38, can be detected in theplasma of animals and humans minutes after the administration of CPT-11(Stewart, et al. 1997. Cancer Chemother. Pharmacol. 40:259-265; Kaneda,et al. 1990. Cancer Res. 50:1715-1720; Rowinsky, et al. 1994. CancerRes. 54:427-436), suggesting that a CE enzyme present in either serum ortissues can convert the camptothecin analog to its active metabolite.When CPT-11 is administered to humans, typically less than 5% of thedrug is converted to SN-38, which is in contrast to mice where greaterthan 50% of the drug is hydrolyzed to SN-38 within the first hour ofdosing (Morton, et al. 2005. Cancer Chemother. Pharmacol. 56:629-636).This may be due to either the different levels of CEs expressed in thesespecies, or the proficiency of drug hydrolysis of the different CEs.

Since the activation of CPT-11 in humans is relatively inefficient, CEenzyme prodrug therapy approaches have been examined. For example, anenzyme/prodrug therapy approach using a rabbit liver CE (rCE) which ismuch more efficient at drug activation has been developed (Danks, et al.1999. Cancer Res. 5:917-924; Meck, et al. 2001. Cancer Res.61:5083-5089; Potter, et al. 1998. Cancer Res. 52:2646-2651; Wagner, etal. 2002. Cancer Res. 62:5001-5007; Wierdl, et al. 2001. Cancer Res.61:5078-5082). Using the rCE in therapy, increased sensitivity to CPT-11was accomplished in human tumor cells grown in culture and in xenograftsin immune-deprived mice. It has been suggested, however, that theapplication of the rCE to human therapy may be limited due to thepotential immunogenicity of the lagomorph protein. Human CE enzymes havebeen examined, but, in vitro studies suggest that human intestinal CE(hiCE) is not as efficient at drug activation when compared to rCE(Humerickhouse, et al. 2000. Cancer Res. 60:1189-1192; Khanna, et al.2000. Cancer Res. 60:4725-4728). Additionally, while sensitization ofcells to CPT-11 expressing hiCE has been reported (e.g., Khanna, et al.2000. Cancer Res. 60:4725-4728), studies indicate that the levels andduration of hiCE expressed are much lower than can be achieved with rCE.

The development of new effective treatment strategies for cancer isdependent upon the availability of specific drug screening assays.Specific drug screening assays can involve isolated target tissuemodels, i.e., isolated heart, ileum, vasculature, or liver from animalssuch as rabbits, rats, and guinea pigs, wherein the target tissue isremoved from the animal and a selected activity of that target tissue ismeasured both before and after exposure to the candidate drug. Anexample of a selected activity measured in drug screening assays toidentify new cancer agents is the activity of enzymes such astopoisomerase I or II, which are known to modulate cell death. Suchassays can also be used to screen for potential prodrugs which areconverted to the active metabolite in selected tissues or to identifyselected tissues capable of converting prodrug to its active metabolite.

However, any molecular event that is shown to be modified by a novelclass of compounds can be developed as a screening assay for selectionof the most promising compounds for therapeutic development. In fact,the idea of modulating cells at the genomic level has been applied tothe treatment of diseases such as cancer. Gene therapy for treatment ofcancer has been the focus of multiple clinical trials approved by theNational Institutes of Health Recombinant DNA Advisory Committee, manyof which have demonstrated successful clinical application (Hanania, etal. 1995. Am. Jour. Med. 99:537-552; Johnson, et al. 1995. J. Am. Acad.Derm. 32(5):689-707; Barnes, et al. 1997. Obstetrics and Gynecology89:145-155; Davis, et al. 1996. Current Opinion in Oncology 8:499-508;Roth and Cristiano 1997. J. Natl. Canc. Inst. 89(1):21-39). Tospecifically target malignant cells and spare normal tissue, cancer genetherapies must combine selective gene delivery with specific geneexpression, specific gene product activity, and, possibly, specific drugactivation. Significant progress has been made in recent years usingboth viral (retrovirus, adenovirus, adeno-associated virus) and nonviral(liposomes, gene gun, injection) methods to efficiently deliver DNA totumor sites. Genes can be transfected into cells by physical means suchas scrape loading or ballistic penetration, by chemical means such ascoprecipitation of DNA with calcium phosphate or liposomalencapsulation; or by electro-physiological means such aselectroporation. The most widely used methods, however, involvetransduction of genes by means of recombinant viruses, taking advantageof the relative efficiency of viral infection processes. Current methodsof gene therapy involve infection of organisms withreplication-deficient recombinant viruses containing the desired gene.The replication-deficient viruses most commonly used includeretroviruses, adenoviruses, adeno-associated viruses, lentiviruses andherpes viruses. The efficacy of viral-mediated gene transfer canapproach 100%, enabling the potential use of these viruses for thetransduction of cells in vivo.

Adenovirus vector systems in particular have several advantages. Theseinclude the fact that non-dividing cells can be transduced; transducedDNA does not integrate into host cell DNA, thereby negating insertionalmutagenesis; the design of adenoviral vectors allows up to 7 kb offoreign DNA to be incorporated into the viral genome; very high viraltiters can be achieved and stored without loss of infectivity; andappropriate plasmids and packaging cell lines are available for therapid generation of infectious, replication-deficient virus (Yang 1992.Crit. Rev. Biotechnol. 12:335-356). The effectiveness ofadenoviral-mediated delivery of genes into mammalian cells in cultureand in animals has been demonstrated.

To increase the specificity and safety of gene therapy for treatment ofcancer, expression of the therapeutic gene within the target tissue mustalso be tightly controlled. For tumor treatment, targeted geneexpression has been analyzed using tissue-specific promoters such asbreast, prostate and melanoma specific promoters and tumor-specificresponsive promoters such as carcinoembryonic antigen, HER-2/neu,Myc-Max response elements, DF3/MUC (Dachs, et al. 1997. Oncol. Res.9:313-25). For example, the utility of herpes simplex virus thymidinekinase (HSV-TK) gene ligated with four repeats of the Myc-Max responseelement, CACGTG, as a gene therapy agent for treatment of lung cancerwith ganciclovir was examined in c-, L- or N-myc-overexpressing smallcell lung cancer (SCLC) cell lines (Kumagai, et al. 1996. Cancer Res.56(2):354-358). Transduction of the HSV-TK gene ligated to this CACGTGcore rendered individual clones of all three SCLC lines more sensitiveto ganciclovir than parental cells in vitro, thus suggesting that aCACGTG-driven HSV-TK gene may be useful for the treatment of SCLCoverexpressing any type of myc family oncogene. Additional experimentswith c-myc have focused on the use of the ornithine decarboxylase (ODC)promoter gene. Within the first intron of the ODC gene are two CACGTG “Eboxes” that provide binding sites for the c-myc protein when bound toits partner protein known as max. Mutation of the E box sequence resultsin the inability of c-myc to transactivate the ODC promoter. Previousreports indicate that reporter constructs containing the ODC promoterfused upstream of the chloramphenicol acetyltransferase gene immediatelyadjacent to the second exon were activated in cells that overexpressc-myc (Bello-Fernandez, et al. 1993. Proc. Natl. Acad. Sci. USA90:7804-7808). In contrast, transient transfection of promoterconstructs in which the E boxes were mutated (CACGTG to CACCTG)demonstrate significantly lower reporter gene activity. These datasuggest that it is possible to activate transcription of specific genesunder control of the c-myc responsive ODC promoter. In the case ofN-myc, N-myc protein is a basic helix-loop-helix (BHLH) protein that candimerize with proteins of the same class. N-myc dimerizes with the BHLHprotein max to form a complex that binds to the CACGTG motif present ingene promoters, such as ODC, resulting in transactivation and expressionof specific genes containing this sequence (Lutz, et al. 1996. Oncogene13:803-812). Studies in a neuroblastoma cell line and tumors have shownthat binding of N-myc to its consensus DNA binding sequence correlateswith N-myc expression, data that indicate that the level of N-myc inneuroblastoma cells is a determining factor in expression of proteinsunder control of promoters containing the CACGTG sequence (Raschella, etal. 1994. Cancer Res. 54:2251-2255). Inhibition of expression of thec-myc gene via antisense oligonucleotides as a means for inhibitingtumor growth has also been disclosed (Kawasaki, et al. 1996. Artif.Organs 20(8):836-48).

In the present invention, a mutant human CE has been developed that canactivate CPT-11 as efficiently as CE from rabbit liver (rCE).Polynucleotides encoding this mutated human carboxylesterase enzyme oractive fragments thereof and polypeptides encoded thereby which arecapable of metabolizing the chemotherapeutic prodrug CPT-11 to activedrug SN-38 are disclosed. This mutant human carboxylesterase enzyme alsohas potential applications to treatments involving other potentialsubstrates such as certain drugs of abuse (e.g., cocaine and heroin) andcertain types of chemical weapons (e.g., organophosphates). It has alsobeen found that compositions comprising a polynucleotide of the presentinvention and a disease-specific responsive promoter can be delivered toselected tumor cells to sensitize the tumor cells to thechemotherapeutic prodrug CPT-11, thereby inhibiting tumor cell growth.

SUMMARY OF THE INVENTION

The present invention relates to polynucleotides encoding a mutant humancarboxylesterase capable of metabolizing a chemotherapeutic prodrug andinactive metabolites thereof to active drug. Polypeptides encoded bythese polynucleotides are also embraced by the present invention as arevectors containing these polynucleotides and host cells harboringvectors which express a mutant human carboxylesterase.

The present invention is also a composition containing a polynucleotideencoding a mutant human carboxylesterase and a disease-specificresponsive promoter.

A method for sensitizing tumor cells to a chemotherapeutic prodrug isalso provided. This method involves transfecting selected tumor cellswith a composition containing a polynucleotide encoding a mutant humancarboxylesterase and a disease-specific responsive promoter of theselected tumor cells.

The present invention is also a method of inhibiting growth of selectedtumor cells which includes sensitizing selected tumor cells to achemotherapeutic prodrug metabolized to active drug by a mutant humancarboxylesterase and administering a chemotherapeutic prodrug.

The present invention also relates to a method of purging bone marrowcells of tumor cells. This method involves removing bone marrow cellsfrom a patient and contacting the bone marrow cells with the mutanthuman carboxylesterase of the present invention and a chemotherapeuticprodrug.

Methods for treating drug addiction and facilitating the metabolism ofan organophosphate with the mutant human carboxylesterase of the presentinvention are also provided as are drug screening assays foridentification of compounds activated by a mutant humancarboxylesterase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts rCE and hCE1 amino acid and nucleic acid sequences. FIG.1A shows the amino acid sequence alignment of residues 356-371 and448-465 from rCE and hCE1 that form the missing loops from the formerenzyme, and a list of hCE1 mutants that were constructed. Underlinedresidues indicate amino acids that were substituted by mutagenesis. FIG.1B shows the alignment of full-length wild-type hCE1 (SEQ ID NO:1) andrCE (SEQ ID NO:2) proteins with loop regions of FIG. 1A indicated.

FIG. 2 depicts the in vitro stability of hCE1, hCE1m6 and hiCE. Enzymeswere aliquoted in 50 mM Hepes, stored at 20° C. and CE activities weredetermined at various time intervals, up to 11 weeks. Data are expressedas the amount of active CE remaining as compared to day 0.

FIG. 3 depicts the growth inhibition curves for COS7 cells treated withCPT-11.

FIG. 4 depicts growth inhibition curves for U373MG cells (brain tumorcell line) expressing either the intracellular (u373IRESmut6) or thesecreted form (U373IRESmut6C) of hCE1mut6, which were treated withCPT-11.

FIG. 5 depicts growth inhibition curves with CPT-11 treatment inuntransfected U373MG cells incubated with the media harvested controlcells (pIRESneo) or cells expressing the secreted form of hCE1mut6(pIRESmut6C).

FIG. 6 shows growth inhibition curves for U373MG cells transduced withAdVC, ADCMVrCE, or ADCMVhCE1m6, following treatment with CPT-11. TheIC₅₀ values for these cells with CPT-11 are 26.8 μM, 0.3 μM and 0.04 μM,respectively.

DETAILED DESCRIPTION OF THE INVENTION

CPT-11 is a promising anti-cancer prodrug, that when given to patients,is converted to its active metabolite SN-38 by a human carboxylesterase.However, the wild-type human enzyme is relatively inefficient and lessthan 5% of the prodrug is metabolized to SN-38 (Rivory, et al. 1997.Clin. Cancer Res. 3:1261-1266). In patients, this prodrug is alsometabolized to APC (Haaz, et al. 1998. Cancer Res. 58:468-472). APC haslittle, if any, active anti-tumor activity and is not converted to anactive metabolite in humans (Rivory, et al. 1996. Cancer Res.56:3689-3694). Accordingly, high concentrations of this prodrug must beadministered to achieve effective levels of active drug in vivo.However, myelosuppression and secretory diarrhea limit the amount ofprodrug that can be administered to patients.

In the present invention, a method of sensitizing tumor cells to reducethe effective dose of a prodrug required to inhibit tumor cell growth isprovided which involves transfecting selected tumor cells with a mutatedhuman CE encoded by a polynucleotide.

In accordance with the present invention, a polynucleotide is providedwhich encodes a mutated human carboxylesterase capable of metabolizing achemotherapeutic prodrug and inactive metabolites thereof to activedrug. By “polynucleotide” it is meant to include any form of DNA or RNAsuch as cDNA or genomic DNA or mRNA, respectively, encoding this mutatedhuman enzyme or an active fragment thereof which are obtained by cloningor produced synthetically by well-known chemical techniques. DNA can bedouble- or single-stranded. Single-stranded DNA can include the codingor sense strand or the non-coding or antisense strand. Thus, the termpolynucleotide also includes polynucleotides which hybridize understringent conditions to the above-described polynucleotides. As usedherein, the term “stringent conditions” means at least 60% homology athybridization conditions of 60° C. at 2×SSC buffer. In a particularembodiment, the polynucleotide is a mutant human cDNA or a homologoussequence or fragment thereof which encodes a polypeptide having similaractivity to that of rabbit liver CE enzyme. Due to the degeneracy of thegenetic code, polynucleotides of the present invention can also includeother nucleic acid sequences encoding this enzyme and derivatives,variants or active fragments thereof. The present invention also relatesto variants of this polynucleotide which may be naturally occurring,i.e., allelic variants, or mutants prepared by well-known mutagenesistechniques.

In particular embodiments, the mutant human CE protein of the presentinvention contains at least the following mutations in reference to awild-type human CE (set forth herein as SEQ ID NO:1): Leu358Ile,Leu362Met, Met363Leu, Ser364Gly, Phe448Tyr, Gln449Arg, Lys459Arg andinsertion of Gln after Met361 (see FIG. 1A). These mutations, whenintroduced into wild-type human CE produce a polypeptide having similaractivity to that of rabbit liver CE enzyme (SEQ ID NO:2). An exemplarymutant human CE polypeptide containing these mutations is set forthherein as SEQ ID NO:3.

Mutation of the wild-type hCE1 protein (SEQ ID NO:1) can be achievedusing any conventional mutagenesis approach including site-directedmutagenesis methods well-known in the art with nucleic acids encodingwild-type hCE1 protein as a template. An exemplary wild-typehCE1-encoding nucleic acid molecule is set forth herein as SEQ ID NO:4.

The present invention also provides vectors which harbor polynucleotidesof the present invention and host cells which are genetically engineeredwith vectors of the present invention to produce mutant human CE oractive fragments of this enzyme. Generally, any vector suitable tomaintain, propagate or express polynucleotides to produce the enzyme inthe host cell can be used for expression in this regard. In accordancewith this aspect of the invention the vector can be, for example, aplasmid vector, a single- or double-stranded phage vector, or a single-or double-stranded RNA or DNA viral vector. Such vectors include, butare not limited to, chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, bacteriophages, yeastepisomes, yeast chromosomal elements, and viruses such as baculoviruses,papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids and phagemids. Selection of anappropriate promoter to direct mRNA transcription and construction ofexpression vectors are well-known. In general, however, expressionconstructs will contain sites for transcription initiation andtermination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will include a translation initiating codon at thebeginning and a termination codon appropriately positioned at the end ofthe polypeptide to be translated. Examples of eukaryotic promotersroutinely used in expression vectors include, but are not limited to,the CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter. Vectors comprising thepolynucleotides can be introduced into host cells using any number ofwell-known techniques including infection, transduction, transfection,transvection and transformation. The polynucleotides can be introducedinto a host alone or with additional polynucleotides encoding, forexample, a selectable marker. Host cells for the various expressionconstructs are well-known, and those of skill can routinely select ahost cell for expressing the mutated human CE enzyme in accordance withthis aspect of the present invention. Examples of mammalian expressionsystems useful in the present invention include, but are not limited to,the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines, the COS-7line of monkey kidney fibroblasts, and neural stem cells. In the case ofthe use of neural stem cells as a delivery system, U.S. PatentApplication Nos. 20050019313 and 20050169897 provide methods fordevelopment and application of such cells as a delivery system.

The present invention also relates to compositions containing apolynucleotide of the present invention which have been found to beuseful in sensitizing tumor cells to CPT-11 cytotoxicity by combinationtherapy of the prodrug and a CE enzyme. The present invention thusprovides methods for sensitizing tumor cells to a prodrug oncologicagent. In this context, by “sensitizing” it is meant that the effectivedose of the prodrug can be reduced when the compositions and methods ofthe present invention are employed. In a case where the prodrug'stherapeutic activity is limited by the occurrence of significanttoxicities, or dose-limiting toxicities, sensitization of tumor cells tothe prodrug is especially useful.

While rabbit CE (rCE) and human liver CE, (hCE1) are structurallysimilar, demonstrating 81% amino acid identity (Danks, et al. 1999.Clin. Cancer Res. 5:917-924) and only a ˜1.0 Å RMSD variation over 455residues of the α-carbon trace (Danks, et al. 1999. Clin. Cancer Res.5:917-924; Bencharit, et al. 2002. Nat. Struct. Biol. 9:337-342;Bencharit, et al. 2003. Chem. Biol. 10:341-349), the latter enzyme isvery inefficient at CPT-11 activation (100- to 1000-fold lower than rCE.In a comparison of the x-ray crystal structures of rCE and hCE1, twoloops in rCE (amino acids 356-371 and 450-465; see FIGS. 1A and 1B) wereidentified that were apparently missing from the crystal structure ofhCE1. These loops formed the entrance to the active site of the protein,and it was assumed that the structures of these domains could not bedetermined due to enhanced flexibility and thermal motion. It has beenreported that the substrate specificity of CEs is in part determined bythe constraints enforced by loops that surround the active site entrance(Wadkins, et al. 2001. Mol. Pharmacol. 60:355-362). Thus, the more rigidstructure of these domains in hCE1 protein may significantly impact theability of the protein to activate CPT-11. A series of hCE1 mutants wasdeveloped that contained multiple amino acid substitutions in these loopregions such that the sequence in these regions was identical to rCE.The five mutants constructed are listed in Table 1 (hCE1mut2 throughhCE1mut6). The mutants were inserted into a plasmid, pCIneo, which wasobtained from Promega (Madison, Wis.). The cDNAs for the wild-typeenzymes are found in Genbank: hCE1, Genbank Accession No. M73499 (SEQ IDNO:4); rCE, Genbank Accession No. AF036930 (SEQ ID NO:5).

TABLE 1 Name Mutations pCIhCE1mut2 L362M, M363L pCIhCEmut3 L362M, M363L,K459R pCIhCEmut4 L362M, M363L, K459R, F448Y, Q449R pCIhCEmut5 L362M,M363L, K459R, F448Y, Q449R, L358I, S364G pCIhCEmut6 L362M, M363L, K459R,F448Y, Q449R, L358I, S364G, Q362 insertion

Site-directed mutagenesis was used to produce the hCE1 mutants. Allmutants were subjected to DNA sequence analysis to confirm the identityof the clones. The mutant cDNAs were expressed from pCIneo in Cos7cells. In addition to the hCE1 mutants listed in Table 1, a plasmidcontaining wild-type hCE1 cDNA was constructed (pCIhCE1), and mutant rCEplasmids were also constructed as listed in Table 2.

TABLE 2 Name Details pCIrCEmut2 I357L, R459K pCIrCEmut3 I357L, R459K,M362L pCIrCEmut4 I357L, R459K, M362L, L363M, Q364S, Y448F, R449QpCIrCEmut5 I357L, R459K, M362L, L363M, Q364S, Y448F, R449Q, Q361deletion

The CE mutants were then tested to characterize their ability toactivate CPT-11. To assess CPT-11 activation by the hCE1 mutantproteins, the cDNAs had been ligated into the expression vector pCIneoand transiently produced the protein in Cos7 cells. Whole cell sonicateswere monitored for CE activity. Results were expressed as nmoleso-nitrophenol produced per minute per milligram of total protein. Tocorrect for differences in CE expression within transfected cells, theenzyme activity values were corrected for the level of immunoreactive CEprotein as determined using a western bolt analysis. The results of theanalysis are shown in Table 3. Following transfection with allhCE1-containing plasmids, all extracts demonstrated CE activity, and allhad similar levels of CE protein as determined by western blot analysisusing an anti-hCE1 antibody. However, only hCE1mut6 was capable ofconverting CPT-11 to SN-38.

TABLE 3 CE Activity CPT-11 Converting Plasmid (nmol/min/mg) Activity(pmol/hr/mg CE) pCIneo  5.5 ± 0.1 Not detected (ND) hCE1 119.0 ± 5.0  NDhCE1mut2 267.2 ± 65.2 ND hCE1mut3 158.4.5 ± 20.2  ND hCE1mut4 450.8 ±45.7 ND hCE1mut5 369.5 ± 26.8 ND rCE (wild-type) 332.7 ± 17.2 41  hCE1mut6 202.8 ± 10.3 40.5

To directly compare the ability of hCE1 m6 and rCE to activate CPT-11,both proteins were expressed in COS-7 cells and the ability of extractsto hydrolyze the drug was assessed. In these studies, the levels ofCPT-11 activation were corrected for the amounts of CE protein in thecell extracts by western analysis. This was necessary since it wasunclear whether the mutations would influence the ability of the CEs tometabolize o-NPA, which is used as a measure of enzyme activity. Asindicated in Table 3, hCE1m6 and rCE were essentially equally efficientat CPT-11 hydrolysis.

Since mutation of the loop domain in hCE1 with residues present in rCEincreased the ability of the wild-type hCE1 protein to hydrolyze CPT-11,it was postulated that the converse mutations (i.e., hCE1 residuessubstituted into rCE) would reduce the ability of rCE to activateCPT-11. This panel of mutant rCE proteins is listed in Table 2. Theability of these mutants to convert CPT-11 to SN-38 was then assessedusing the methods described above. The results are listed in Table 4.

TABLE 4 CE Activity CPT-11 Converting Plasmid (nmol/min/mg CE) Activity(pmol/hr/mg CE) rCE (wild-type) 243.1 ± 17.0 75.6 rCEmut2 333.1 ± 7.0 52.5 rCEmut3 170.7 ± 11.0 47.4 rCEmut4 141.4 ± 1.8  41.7 rCEmut5 219.9 ±5.8  11.5 hCE1 (wild-type) 329.2 ± 11.6 Not Detected

Substitution of the amino acids present within loops of rCE withcorresponding residues from hCE1 resulted in a gradual reduction in theability of the protein to hydrolyze CPT-11. A mutant that containedeight amino acid changes, rCEmut5, was 7-fold less efficient at drugactivation than wild-type rCE. Mutants with intermediate numbers ofamino acid substitutions demonstrated intermediate abilities tohydrolyze CPT-11. These data demonstrate that there was a gradient ofcatalytic activity, where the mutant with the smallest number ofsubstitutions demonstrated the lowest reduction in ability to hydrolyzeCPT-11.

To directly compare the abilities of the mammalian CEs to hydrolyzeCPT-11, a series of biochemical studies were carried out using thepurified proteins in vitro. These experiments determined the K_(m),V_(max), k_(cat) and k_(cat)/K_(m) values for the different enzymes. Asindicated in Table 5, hCE1m6 was ˜70-fold more efficient at CPT-11hydrolysis as compared to the hCE1. In addition, hCE1m6 was almost aseffective at CPT-11 hydrolysis as hiCE. It should be noted that bothhiCE and hCE1m6 were less efficient at drug activation than rCE, howeversince hiCE has been effectively used in prior enzyme/prodrug therapyapproaches (Oosterhoff, et al. 2005. Br. J. Cancer 92:882-887;Oosterhoff, et al. 2002. Br. J. Cancer 87:659-664; Oosterhoff, et al.2005. Gene Ther. 12:1011-1018; Oosterhoff, et al. 2003. Mol. Cancer.Ther. 2:765-771), hCE1m6 is expected to be efficacious in CPT-11activation in vivo.

TABLE 5 Ratio k_(cat)/K_(m) Km Vmax k_(cat)/K_(m) as compared Enzyme(μM) (nmol/min/mg) (mM⁻¹ min⁻¹) to hCE1 hCE1^(a) 82.8 ± 9.6   0.36 ±0.017 0.28 1 hCE1m6 6.25 ± 0.59 2.11 ± 0.06 19.8 71 rCE^(a) 6.20 ± 0.63 18 ± 0.9 180.0 650 hiCE 3.35 ± 0.34 1.49 ± 0.04 25.2 91 ^(a)Data takenfrom Wadkins, et al. 2001. Mol. Pharmacol. 60: 355-362 (29).

In previous biochemical experiments, it was noted that in vitro purifiedhiCE was much less stable than hCE1. Therefore, the loss of CE activitywas evaluated in preparations of these proteins, as well as hCE1m6 thathad been stored at room temperature. In these studies, purified proteinwas aliquoted in 50 mM Hepes pH7.4, and enzyme activity was determinedover a period of 12 weeks. As indicated in FIG. 2, hiCE was the leaststable protein, losing 50% of its activity by ˜30 days. In contrast,both hCE1m6 and hCE1 were relatively stable under these conditions withpredicted half-lives of 129 and 440 days, respectively.

In in vivo studies, it was observed that expression of hiCE followingplasmid-mediated transfection was frequently lower than that seen withrCE or hiCE. Therefore, U373MG cells expressing hiCE, hCE1 or hCE1m6were developed using the plasmid pIRESneo, and CE expression in thederived lines was monitored over an extended time period. Sinceexpression of the transgene from this expression vector is co-regulatedwith the neo gene via an IRES sequence, selection with similarconcentrations of G418 should result in cell lines that expressapproximately equal levels of recombinant protein. However, the averagelevels of CE activity in U373hiCE, U373hCE1 and U373hCE1m6 cells were287.6±76.6, 1077.1±77.5, and 466.5±52.6 nmoles/min/mg, respectively.Since high level expression of the prodrug-activating protein would benecessary for effective application of enzyme/prodrug therapy, hiCE maynot be the best CE for activating CPT-11.

The sensitivity of mammalian cells to CPT-11 following expression ofhCE1mut6 was then determined by constructing cell growth inhibitioncurves for human tumor cells expression the mutant protein. Cos7 cellstransiently transfected with hCE1mut6, as described above, werecontacted with increasing concentrations of CPT-11 in order to determinethe concentration of drug required to inhibit cell growth by 50% (IC₅₀).FIG. 3 shows that cells expressing the hCE1mut6 protein were equally assensitive to CPT-11 as cells expressing rCE. Both populations of cellshad similar IC₅₀ values for CPT-11, approximately 12 to 20 micromolar.This value was 5-fold less that the IC₅₀ value for cells expressingwild-type hCE1 (IC₅₀=77.1 micromolar).

Experiments were also performed in human brain tumor cells (U373MG)where the growth of the cells was examined in cells that expressed themutant form of the human CE enzyme. As indicated in Table 6, cellsexpressing hCE1m6, hiCE or rCE were sensitized to CPT-11 due tointracellular conversion of the drug to SN-38. Cells expressing hCE1were not sensitized, consistent with the lack of CPT-11 hydrolysisobserved in the biochemical studies. Furthermore, U373MG cellsexpressing hCE1m6 were equally as sensitive to CPT-11 as cellsexpressing rCE, with IC₅₀ values ranging from 0.18-0.40 μM. These valueswere ˜18- to 86-fold less than cells expressing wild-type hCE1 (IC₅₀value=15.5 μM). These results indicate that hCE1mut6 can efficientlyconvert CPT-11 to SN-38 intracellularly and sensitize cells to the drug.

TABLE 6 Enzyme CE activity CPT-11 Fold decrease Cell line Adenovirusexpressed (nmol/min/mg ± SD) IC₅₀ (μM) in IC₅₀* U373IRES — None 10.0 ±0.3 24.0 — U373hCE1 — hCE1 1016.4 ± 45.5  15.5 — U373hiCE — hiCE 408.2 ±5.6  0.84 18 U373rCE — rCE 601.0 ± 20.5 0.40 39 U373hCE1m6 — hCE1m6437.3 ± 37.5 0.18 86 U373MG AdVC None 10.0 ± 0.5 26.8 — AdCMVrCE rCE1076.8 ± 67.3  0.3 89 AdCMVhCE1m6 hCE1m6 5999.8 ± 162.5 0.04 670  Rh30AdVC None  4.6 ± 0.3 64.3 — AdCMVrCE rCE 665.6 ± 52.0 3.4 29 AdCMVhCE1m6hCE1m6 2757.6 ± 87.5  2.0 32 SK-N-As AdVC None  6.9 ± 0.3 31.7 —AdCMVrCE rCE 2150.3 ± 105.9 0.6 53 AdCMVhCE1m6 hCE1m6 6225.0 ± 113.4 0.563 *value as compared to U373MGhCE1 or cell line + AdVC.

Experiments were also performed to determine whether cells expressing asecreted form of hCE1m6 produced media that had CE activity. U373MGcells expressing either the intracellular (u373IRESmut6) or the secretedform (U373IRESmut6C) of hCE1mut6 were treated with CPT-11. There was anapproximate 130-fold reduction in the IC₅₀ value for CPT-11 in the cellsexpressing hCE1mut6 (either form). However, cells expressing thesecreted form of the protein had an intermediate IC₅₀ for CPT-11 (7.08μM) because the intracellular levels of the CE were lower due to thesecretion into the culture media (FIG. 4).

Growth inhibition of untransfected U373MG cells was also examinedfollowing incubation with the media harvested control cells (pIRESneo)or cells expressing the secreted form of hCE1mut6 (pIRESmut6C) andCPT-11. Since the enzyme was secreted into the media and able toactivate CPT-11, the active metabolite SN-38 was produced, resulting incytotoxicity to cells that did not express the mutant CEs. This effectwas not observed with the control media. The difference in the IC₅₀values was approximately 150-fold (FIG. 5). These results are indicativeof a bystander or collateral effect.

E1A, E3-deleted replication-deficient adenovirus expressing hCE1m6 werealso generated and the ability of this vector to sensitize cells toCPT-11 was determined. As shown in Table 6, in all human tumor celllines, expression of hCE1m6 significantly decreased the CPT-11 IC₅₀values as compared to vector transduced cells. In SK—N—As and Rh30 celllines, the CPT-11 sensitivity was comparable to cells transduced withAdCMVrCE (adenovirus containing the rabbit liver CE cDNA), with IC₅₀values of 2 nM and 0.6 nM, respectively. However, the human astrocytomacell line U373MG, was 7.5-fold more sensitive to CPT-11 afterAdCMVhCE1m6 transduction, than after exposure to AdCMVrCE. This resultedin an overall reduction in the IC_(H) for CPT-11 of ˜670-fold for U373MGcells (FIG. 6), the greatest sensitization observed using thisenzyme/prodrug approach. Overall, these results indicate thatAdCMVhCE1m6 can sensitize cells to CPT-11 as effectively as AdCMVrCE,and that the former vector should be suitable for enzyme/prodrug therapywith this drug.

Thus, one embodiment of the present invention embraces transfectingselected tumor cells with the polynucleotide of the present invention,which expresses mutant human CE. The polynucleotide can expressed via awell-known promoter such as the CMV promoter or, more preferably, via adisease-specific responsive promoter which specifically targets theselected tumor cells. Targeted gene expression in tumor cells has beenachieved using disease-specific responsive promoters such ascarcinoembryonic antigen, HER-2/neu, Myc-Max response elements, andDF3/MUC. Thus, a composition containing the mutant CE disclosed hereinand a disease-specific responsive promoter such as these can be used totransfect and sensitize tumor cells containing the disease-specificresponsive promoter. Accordingly, the present invention provides a meansfor exploiting tumor-specific expression associated with adisease-specific responsive promoter to provide for selective therapy oftumors.

Since myc expression is deregulated in a wide variety of human tumors,myc is an attractive target for chemotherapeutics. No known drugspecifically interacts with either the c-myc or N-myc protein. However,cells overexpressing a myc oncogene can be targeted with compositions ofthe present invention containing a polynucleotide of the presentinvention under the control of a myc specific promoter. Thus, using thepresent invention the tumor-specific overexpression of c-myc and N-myccan be exploited to produce selective killing with a chemotherapeuticagent.

In vivo efficacy of the CE of the present invention to sensitize tumorcells to CPT-11 is examined in different types of tumor cells as well asin a mouse model to demonstrate that the mutant human CE of the presentinvention is capable of sensitizing cells to the growth inhibitoryeffects of CPT-11.

For example, the ability of the mutant human CE, hCE1mut6, to sensitizeRh30 rhabdomyosarcoma human tumor cells grown as xenografts inimmune-deprived mice can be examined. This is a well-acceptedpreclinical model, where expression of the transfected cDNA for hCE1mut6can first be established. Following establishment of tumors in theanimals, treatment with CPT-11 begins. Tumor growth is examined andcompared in animals treated with CPT-11 when the tumor cells had beentransfected with the mutant human CE versus the growth seen in tumorcells not transfected with this mutant human CE, or those transfectedwith other CE forms, such as rCE. The in vivo effects of the hCE1mut6protein can also be examined in a model such as mice with human U373glioblastoma xenografts, where the xenografts express hCE1mut6. Again,the sensitivity of these tumor cells to CPT-11 can be compared toxenografts transfected with a control plasmid.

The data described herein support the use of the combination ofpolynucleotide encoding a mutant CE of the present invention and CPT-11to reduce the amount of CPT-11 needed to produce inhibition of tumorcell growth, or to sensitize the tumor cells to CPT-11. These data canalso be used to support the use of the present invention to allow fordecreased dosage with CPT-11 in cancer patients, thus reducing thelikelihood of dose-limiting toxicity.

The present invention thus also relates to a method for treating cancerwith reduced side effects. In one embodiment, a polynucleotide of thepresent invention is inserted into a viral vector using a gene transferprocedure. Preferred viral vectors include, but are not limited to,retroviral, adenoviral, herpesvirus, vaccinia viral and adeno-associatedviral vectors. In this embodiment, it is preferred that the vectorfurther include a disease-specific responsive promoter. The vectors canthen be injected into the site of tumor removal along with systemicadministration of a prodrug such as CPT-11 to inhibit the recurrence oftumors due to residual tumor cells present after surgical resection of atumor.

Alternatively, the viral vector can be used to purge bone marrow ofcontaminating tumor cells during autologous transplant. Bone marrowpurging via a viral vector such as adenovirus which expresses a CE ofthe present invention is performed ex vivo. Efficiency of removal ofcontaminating tumor cells is determined by PCR assays of purged samples.Data indicate that the method of the present invention is applicable toan animal model for purging bone marrow of neuroblastoma cells such asthat described in Example 6. Methods for preparation of the vectors,modes of administration, and appropriate doses of prodrug are well-knownto those of skill in the art. Other methods of gene delivery such aschemical and liposome-mediated gene transfer, receptor-mediated DNAuptake, and physical transfer by gene guns or electroporation can alsobe employed.

Another method for delivering CEs to selected tumor cells involvesantibody-directed enzyme prodrug therapy (ADEPT). In this method, humantumors are targeted by conjugation of tumor-specific marker antibodywith a molecule such as hCE1mut6. Cellular internalization of thecomplex and release of active CE is achieved, leading to CPT-11activation that is specific for cells expressing the marker antigen.Since the array of marker molecules expressed upon the cell surface isdifferent for each tumor type, markers specific for each targeted tumortype can be selected as appropriate. Similarly, the use ofavidin-biotin-conjugated molecules to target tumor cells (Moro, et al.1997. Cancer Res. 57:1922-1928) is also applicable for localization ofthe CE protein of the present invention to the cell surface followed bydrug activation at the targeted cell.

Liver CE is localized in the endoplasmic reticulum. Removal of the sixN-terminal amino acids results in secretion of active protein into theextracellular milieu. Both the secreted and the endoplasmicreticulum-localized protein can convert CPT-11 to SN-38; therefore, thepotential exists for a bystander effect from cells expressing thesecreted enzyme. A similar bystander effect has been demonstrated forother enzyme/prodrug combinations, such as HSVtk and ganciclovir(Dilber, et al. 1997. Cancer Res. 57:1523-1528), and results inincreased cytotoxicity. Extracellular activation of CPT-11 may result inmore efficient eradication of MRD in that uninfected neighboring tumorcells would be killed by exogenously produced SN-38. Gene therapyprotocols with a secreted CE in combination with CPT-11 can therefore bemore appropriate for the elimination of residual tumor tissue.Accordingly, it may be preferred to use a fragment of a polynucleotideencoding a polypeptide which is secreted, i.e., a polypeptide lackingthe six N-terminal amino acid residues. An exemplary mutant human CEprotein lacking the six N-terminal amino acid residues is set forthherein as SEQ ID NO:16. Additionally, recent reports indicate that thetethering of drug activating enzymes to the extracellular cell surfacecan result in anti-tumor activity in human tumor xenografts whencombined with appropriate prodrug (Marais, et al. 1997. Nature Biotech.15:1373-1377). A tethered enzyme generates a local bystander effectsince the protein is not free to circulate in the plasma. Attachment ofa CE of the present invention to the cell surface should result in localextracellular activation of CPT-11 to SN-38 and enhance local cell kill.Purging bone marrow of contaminating tumor cells will be accomplished byan intracellular enzyme, whereas eradication of MRD is better achievedby an enzyme that activates CPT-11 at an extracellular location.

Another aspect of the present invention that is contemplated is theapplication of the mutant human CE enzyme in the treatment of drugaddiction. This use is contemplated based on work that has shown theability of human CE to catalyze the hydrolysis of drugs such as heroinand cocaine (Redinbo, et al. 2003. Biochemi. Soc. Trans. 31:620-624;Bencharit, et al. 2003. Nat. Struct. Biol. 10:349-356). As such, the useof the instant mutant human CE enzyme to affect drug metabolism can beexploited as a method for treatment of drug addiction, as would beappreciated by one of skill in the art. In general, this application ofthe mutant human CE enzyme would be in a manner similar to the use ofthe enzyme in treatment of cancer.

In yet another aspect of the present invention that is contemplatedbased on the results described herein, the mutant human CE enzyme couldbe used in a method for metabolizing certain types of chemical weaponsagents, specifically organophosphate compounds. It has been shown thatorganophosphates can be efficiently hydrolyzed by human CE after asingle point mutation in its active site (Redinbo, et al. 2003.Biochemi. Soc. Trans. 31:620-624). In this regard, the enzyme of thepresent invention can be administered to a subject in need of treatment,e.g., a subject exposed to or suspected of being exposed to a chemicalweapon agent, and used as a preventative treatment against the toxicityresulting from exposure to organophosphates.

The CE of the present invention cleaves the COOC bond present as anester linkage in CPT-11 to generate SN-38. Since this enzyme can alsocatalyze the activation of other compounds that contain such a linkage,the present invention also provides assays for screening for compoundsthat contain this and related moieties. In one embodiment, the assay ofthe present invention is conducted in a cell system using, for example,yeast, baculovirus, or human tumor cell lines. In this embodiment,compounds activated by CE are identified and assessed for anticanceractivity by growth inhibition or clonogenic cell survival assays usingcells expressing or lacking a CE of the present invention.Alternatively, compounds can be screened in cell-free assays using a CEof the present invention isolated from host cells expressing thisenzyme. In this embodiment, the ability of the enzyme to cleave a COOCester linkage of a candidate compound is measured directly in a standardenzyme assay buffer system containing a CE of the present invention.Known concentrations of candidate compounds can be added to assay tubescontaining a biological buffer such as HEPES at pH 7.4 and the enzymeand incubated at 37° C. for a selected amount of time. The reaction isthen terminated by addition of methanol. The assay tubes are thencentrifuged and the supernatant analyzed for the presence of cleavedcompound fragment. Analysis of the supernatant can be performed by anynumber of well-known techniques including, but not limited to,spectrofluorometric analysis, high pressure liquid chromatography ormass spectrometry.

Compounds which can be screened in accordance with the instant assayinclude small organic compounds as well as derivatives or analogs ofknown compounds which contain the COOC ester linkage (e.g., CPT-11).Compounds identified in these screening assays as potential anticancerprodrugs may require chemical modification for optimize their anti-tumoractivity.

The following non-limiting examples are provided to further illustratethe claimed invention.

Example 1 Materials and Methods

Cell Lines, Plasmids and Adenoviral Vectors. Cell lines were grown in10% fetal bovine serum and 2 mM glutamine in an atmosphere of 10% CO₂ at37° C.

Plasmids containing the cDNAs encoding hCE1, hiCE and rCE are known inthe art (Danks, et al. 1999. Clin. Cancer Res. 5:917-924; Potter, et al.1998. Cancer Res. 52:2646-2651; Khanna, et al. 2000. Cancer Res.60:4725-4728). The Genbank accession numbers for these sequences areM73499 (Munger, et al. 1991. J. Biol. Chem. 266:18832-18838), Y09616(Schwer, et al. 1997. Biochem. Biophys. Res. Comm. 233:117-12) andAF036930 (Potter, et al. 1998. Cancer Res. 52:2646-2651), respectively.All of the plasmids, cell lines and adenoviral vectors used in thesestudies are listed in Table 7.

TABLE 7 Name Description Details PCIneo* Mammalian Obtained from Promegaexpression vector pCIhCE1* pCIneo containing From Danks, et al. 1999.Clin. wild-type hCE1 cDNA Cancer Res. 5: 917-924 pCIrCE* pCIneocontaining From Potter, et al. 1998. Cancer wild-type rCE cDNA Res. 52:2646-2651 pCIhCE1m2* pCIneo containing M363L, L364M mutant hCE1 cDNApCIhCE1m3* pCIneo containing M363L, L364M, K459R mutant hCE1 cDNApCIhCE1m4* pCIneo containing M363L, L364M, K459R, F448Y, Q449R mutanthCE1 cDNA pCIhCE1m5* pCIneo containing M363L, L364M, K459R, F448Y,Q449R, mutant hCE1 cDNA L357I, S365G pCIhCE1m6* pCIneo containing M363L,L364M, K459R, F448Y, Q449R, mutant hCE1 cDNA L357I, S365G, Q361insertion pIRESneo* Mammalian Contains G418 resistance gene expressionvector coupled to an IRES sequence pIRESrCE* pIRESneo From Potter, etal. 1998. Cancer containing rCE cDNA Res. 52: 2646-2651 pIREShCE1*pIRESneo From Danks, et al. 1999. Clin. containing hCE1 cDNA Cancer Res.5: 917-924 pIREShiCE* pIRESneo Expresses hiCE following containing hiCEcDNA transfection and selection with G418 pIREShCE1m6* pIRESneo Containsmutations as listed above containing hCE1m6 cDNA for pCIhCE1m6 COS-7^(#)African green Obtained from the America Type monkey kidney cell lineCulture Collection U373MG^(#) Human astrocytoma Obtained from theAmerica Type cell line Culture Collection U373IRES^(#) U373MGtransfected G418 resistant but lacking with pIRESneo exogenous CEexpression U373hCE1^(#) U373MG transfected U373MG expressing hCE1 withpIREShCE1 U373rCE^(#) U373MG transfected U373MG expressing rCE withpIRESrCE U373hiCE^(#) U373MG transfected U373MG expressing hiCE withpIREShiCE U373hCE1m6^(#) U373MG transfected U373MG expressing hCE1m6with pIREShCE1m6 293^(#) Human embryo Obtained from the America Typekidney cell line Culture Collection Rh30^(#) Rhabdomyosarcoma FromDouglass, et al. 1987. cell line Cytogenet. Cell Genet. 45: 148-155SK-N-AS^(#) Neuroblastoma cell line Obtained from the America TypeCulture Collection AdVC^(†) Adenovirus vector E1, E3-deleted Ad vectorbased upon Ad5 AdCMVrCE^(†) Adenovirus Expresses high levels of rCEunder containing rCE cDNA control of CMV promoter AdCMVhCE1m6^(†)Adenovirus Expresses high levels of hCE1m6 containing hCE1m6 cDNA undercontrol of CMV promoter *plasmid; ^(#)cell line; ^(†)adenovirus.

Analysis of Carboxylesterase Crystal Structures. The x-ray crystalstructures of rCE (PDB 1K4Y; Bencharit, et al. 2002. Nat. Struct. Biol.9:337-342)) and hCE1 (PDB 1MX5; Bencharit, et al. 2003. Chem. Biol.10:341-349; Bencharit, et al. 2003. Nat. Struct. Biol. 10:349-356) wereoverlaid and examined using ICM Pro software (Molsoft, San DiegoCalif.).

Site-Directed Mutagenesis. Site-directed mutagenesis was achieved usinga QuickChange site-directed mutagenesis kit (Stratagene, La Jolla,Calif.) with custom primers designed to produce the desired mutations.All mutants were subjected to DNA sequencing to confirm the identity ofthe clones.

Carboxylesterase Assays. CE activity was determined using aspectrophotometric assay with 3 mM o-nitrophenyl acetate (o-NPA) as asubstrate (Potter, et al. 1998. Cancer Res. 52:2646-2651; Wierdi, et al.2001. Cancer Res. 61:5078-5082; Beaufay, et al. 1974. J. Cell Biol.61:188-200). Data were expressed as nmoles o-nitrophenol produced perminute per milligram of protein. To correct for differences in CEexpression within transfected cells, the enzyme activity values werecorrected for the level of immuno-reactive CE protein as determined fromwestern analyses.

Transfection of Cell Lines. Transient transfection of Cos7 cells wasachieved by electroporation (Potter, et al. 1998. Cancer Res.52:2646-2651). For the generation of stable cell lines, cDNAs wereligated into pIRESneo and U373MG cells were electroporated under similarconditions. Transfectants were selected in media containing 400 μg/ml ofG418. Since the CE cDNA was linked via an internal ribosome entrysequence (IRES) to the neomycin gene, selection of individualG418-resistant clones was not necessary. Routinely, whole cell sonicatesobtained from these pooled populations of cells contained 200-500nmoles/min/mg of CE activity.

CPT-11 Conversion Assays. Conversion of CPT-11 to SN-38 was monitored byincubating cell extracts with 5 μM CPT-11 for 1 hour in 50 mM HepespH7.4 at 37° C. An equal volume of acidified methanol was added toterminate the reactions and particulate matter was removed bycentrifugation at 100,000 g for 5 minutes at 4° C. Concentrations ofboth drugs in the supernatant were then determined by HPLC (Guichard, etal. 1998. Clin. Cancer Res. 4:3089-3094; Morton, et al. 2000. CancerRes. 60:4206-4210).

Protein Purification. Secreted forms of hCE1, rCE and mutant human CEwere expressed in Spodoptera frugiperda Sf9 cells and purified fromserum-free culture media according to known methods (Morton & Potter.2000. Mol. Biotechnol. 16:193-202). For hiCE, an alternativepurification procedure was developed using DEAE chromatography andelution with a pH/salt gradient.

Determination of Kinetic Parameters for Substrate Hydrolysis. K_(m),V_(max) and k_(cat) values for the hydrolysis of CPT-11 by therecombinant purified proteins were determined using standard methods(Wadkins, et al. 2001. Mol. Pharmacol. 60:355-362).

Western Analysis. Cell extracts were separated in 4-20% pre-castSDS-PAGE gels (Invitrogen, Carlsbad, Calif.) and following transfer toImmobilon-P membranes by electroblotting (Matsuidaira. 1990. MethodsEnzymol. 182:602-613), western analysis was performed using knownmethods (Morton & Potter. 1998. J. Pharmacol. Expt. Therap.286:1066-1073). CEs were identified using an anti-peptide antibodyraised against the C-terminal amino acids CEKPPQTEHIEL (SEQ ID NO:15) ofhCE1, and ECL detection (Amersham Life Sciences, Arlington Heights,Ill.). In all experiments, membranes were re-probed with an anti-TFIIDantibody to confirm equal loading, and to correct for any differences intotal protein. The molecular weight of immuno-reactive bands wasdetermined using pre-stained molecular weight protein markers (Pierce,Rockford, Ill.). Densitometric quantitation of CE expression wasperformed using One-Dscan gel analysis software (Scanalytics Inc,Fairfax, Va.).

Construction of adenovirus. Replication-deficient adenovirus expressinghCE1m6 or rCE were constructed using standard protocols (Wierdl, et al.2001. Cancer Res. 61:5078-5082). Multiplicity of infection (moi) wasdefined as the number of plaques produced in 1×10⁶ 293 cells in a totalvolume of 1 ml of media after incubation with virus for 1 hour.Typically for IC₅₀ determinations an moi of 5 was used, however, usingthese conditions in U373MG cells resulted very high levels of transgeneexpression, leading to toxicity, Therefore an moi of 1 was used for thiscell line.

Growth Inhibition Assays. Growth inhibition assays using CPT-11 wereperformed in triplicate in 6 well multiwell plates as previouslydescribed (Wierdl, et al. 2004. Biochemistry 43:1874-1882). Theconcentrations of drug required to inhibit cell growth by 50% (IC₅₀values) were calculated using Prism software (GraphPad Software, SanDiego, Calif.).

Example 2 In vitro Biological Activity of CE

The in vitro activity of rabbit liver CE can be examined in tumor celllines. The growth inhibition of CPT-11 is compared in cells with andwithout hCE1mut6. The cells used can be Rh30 cells (107) that have beenelectroporated with a plasmid DNA or plasmid containing CE cDNA in avolume of phosphate-buffered saline. The cells are plated into 75-cm²flasks in fresh media and G418 added hours following transfection toselect for cells expressing the neo gene and the CE. Cells are grown fora minimum of 10 days before use in growth inhibition experiments.

In one type of assay, CPT-11 is pre-incubated with hCE1mut6 to produceSN-38 prior to exposure of the cells to drug. For example, 0.5 to 5units of hCE1mut6 are incubated with 1 M CPT-11 at 37° C. in DMEM mediumfor 2 hours. Each reaction mixture is filter-sterilized and Rh30 cellsexposed to drug for one hour, at which time the medium is replaced withdrug-free medium containing serum. Enzyme that has been inactivated byboiling for five minutes prior to incubation with drug or CPT-11 towhich no enzyme has been added is used as negative controls. Cells areallowed to grow for three cell doubling times and cell numbers aredetermined.

In another type of growth inhibition assay, Rh30 cells transfected witheither parent plasmid DNA or the plasmid containing the mutant human CEcDNA are exposed to different concentrations of CPT-11. Drug is added totissue culture medium of each of the stably transfected cell lines fortwo hours, after which time the medium is replaced with drug-freemedium. Cells are then allowed to grow for three cell doublings asbefore. Results are expressed as the concentration of drug required toreduce cell growth to 50% of control cells, or IC₅₀.

Example 3 Use of CE in an In Vivo Model for Minimal Residual Disease(MRD)

A xenograft model for MRD has been developed to demonstrate theeffectiveness of the combination of hCE1mut6 and prodrug in theprevention of MRD. In this model, immune-deprived mice, i.e., SCID mice,bearing human NB-1691 xenografts are treated with 10 mg/kg CPT-11 dailyfor 5 days on two consecutive weeks and tumor regression is examined.However, within 4-6 weeks, tumors are palpable in the exact positionwhere the original xenograft was implanted. Since these tumors arisefrom cells that survived the initial cycle of chemotherapy, this modeltherefore mimics results seen in patients following surgical resectionof the primary tumor and subsequent regrowth at the same site.

Experiments are performed in this model to compare the responses of micebearing human Rh30 xenografts as well as xenografts expressing thehCE1mut6 protein. Rh30 rhabdosarcoma xenografts are transfected with aplasmid containing the cDNA for hCE1mut6 and with G418. Expression of CEis confirmed by biochemical assay using the CE substrate o-NPA andmaintained for at least 12 weeks. Two groups of SCID mice are injectedwith the transfected cells subcutaneously into the flanks. A third groupof control mice is injected in identical fashion with Rh30 cells nottransfected with the plasmid. When the tumors reach a size ofapproximately 1 cm³, 2.5 mg CPT-11/kg/day is administered five days eachweek for two weeks (one cycle of therapy), repeated every 21 days for atotal of three cycles (over 8 weeks) to one group of mice injected withthe transfected xenograft cells and the third group of control mice.Tumor regression is examined.

Experiments can also be performed employing U373 glioblastoma cellstransfected with a control plasmid or with a plasmid containing the cDNAfor the hCE1mut6 protein. Expression of CE in the tumor cells isconfirmed by biochemical assay using the substrate o-NPA. Cells areinjected subcutaneously into the flanks of the SCID mice. When tumorsreach approximately 1 cm³ in size, CPT-11 is administered daily for fivedays each week as described above, for three cycles, at a dose of 7.5mg/kg/day. Again, tumor regression is examined.

Example 4 Use of a Human CE/Prodrug Combination to Purge Bone Marrow ofTumor Cells

Intravenous injection of human neuroblastoma NB-1691 tumor cells intoimmune-deprived mice results in the development of widespread metastaticdisease with death occurring on days 36-38. Since both synaptophysin andtyrosine hydroxylase expression are specific for neuroblastoma cells,RT/PCR analysis of these mRNAs can detect tumor cells present in mixedpopulations of cells. Circulating neuroblastoma cells can be detected inthe peripheral blood of these animals 36 days after injection withNB-1691. Studies then determine whether the bone marrow of these sameanimals contains neuroblastoma cells. The success of ex vivo purging ofbone marrow with the hCE1mut6/CPT-11 combination is demonstrated bytransplanting purged bone marrow into lethally irradiated mice. If miceremain disease free for extended periods of time, this indicates thatthe CE/prodrug purging therapy kills neuroblastoma cells in the donormarrow.

Example 5 Treatment of Minimal Residual Disease (MRD) in Humans

The mutant human CE in combination with CPT-11 or other prodrug(s)activated by this enzyme are used to purge bone marrow of residual tumorcells prior to autologous bone marrow transplants to prevent recurrenceof local MRD following removal of bulk tumor by surgery or chemotherapy.Following debulking of the primary tumor, adenovirus containing themutant human CE under the control of a tumor-specific responsivepromoter is applied to the tumor margins at either the time of surgery,by stereotaxic injection, or by implantation of a time-release polymeror other material. Anti-tumor effect of single application at time ofsurgery is compared with the effect produced by repetitive ortime-release use of adenoviral constructs. Adenovirus dose ranges from10⁶ to 10¹⁰ plaque-forming units as has been reported to be effectivefor intratumoral injection of adenovirus (Heise, et al. 1977. NatureMed. 3:639-645). CPT-11 is administered over the next one to six weeksto elicit tumor selective cell kill. Doses and schedules of CPT-11 aredetermined in clinical trials of CPT-11 by itself and in human xenograftmodel systems to produce maximal tumor effect.

Example 6 Purging Bone Marrow of Tumor Cells in Humans

Tumor cells that contaminate bone marrow used for autologous transplantcontribute to relapse of disease. Therefore, the mutant human CE can beused in combination with a suitable prodrug to eradicate tumor cells inmarrow samples to be used for transplant. This approach maintains theviability of hematopoietic cells required for reconstitution. Bonemarrow samples are transduced ex vivo with adenovirus containing themutant human CE cDNA, using a multiplicity of infection (moi) that willinfect 100% of the tumor cells. Typically, a moi of 0.5 to 10 isadequate for tumor cells, while a moi of 100 to 1,000 is required totransduce a majority of hematopoietic progenitor cells. Two daysfollowing adenoviral transduction, cells are exposed for two hours to arange of CPT-11 concentrations, usually varying from 50 nM to 100 μM.Two days after exposure to drug, the marrow sample is harvested andstored for reinfusion into the patient and reconstitution of atumor-free marrow.

1. A method for sensitizing tumor cells to a chemotherapeutic prodrugcomprising transfecting selected tumor cells with a compositioncomprising an isolated polynucleotide comprising a disease-specificresponsive promoter and encoding a mutant human carboxylesterase.
 2. Amethod of inhibiting tumor cell growth comprising: (a) sensitizing tumorcells in accordance with the method of claim 1; and (b) contacting saidsensitized tumor cells with a chemotherapeutic prodrug so that tumorcell growth is inhibited.
 3. The method of claim 2, wherein thechemotherapeutic prodrug is CPT-11.